bims-mitmed Biomed News
on Mitochondrial medicine
Issue of 2024–06–02
25 papers selected by
Dario Brunetti, Fondazione IRCCS Istituto Neurologico
  1. CLUH maintains functional mitochondria and translation in motoneuronal axons and prevents peripheral neuropathy.
  2. Oral mitochondrial transplantation using nanomotors to treat ischaemic heart disease.
  3. Mechanisms and pathologies of human mitochondrial DNA replication and deletion formation.
  4. TBK1 is ubiquitinated by TRIM5α to assemble mitophagy machinery.
  5. Cells dispose of cytoplasmic mitochondrial DNA by nucleoid-phagy.
  6. Targeting the autophagy-NAD axis protects against cell death in Niemann-Pick type C1 disease models.
  7. Mitochondrial iron deficiency triggers cytosolic iron overload in PKAN hiPS-derived astrocytes.
  8. Mitochondrial quality control in human health and disease.
  9. FIP200 phosphorylation regulates late steps in mitophagy.
  10. Spatial detection of mitochondrial DNA and RNA in tissues.
  11. Atg44/Mdi1/mitofissin facilitates Dnm1-mediated mitochondrial fission.
  12. Multifaceted mitochondria in innate immunity.
  13. Mitochondrial defects in sporadic inclusion body myositis-causes and consequences.
  14. ACAD9 treatment with bezafibrate and nicotinamide riboside temporarily stabilizes cardiomyopathy and lactic acidosis.
  15. Neuroglobin overexpression in cerebellar neurons of Harlequin mice improves mitochondrial homeostasis and reduces ataxic behavior.
  16. ISR pathway contribution to tissue specificity of mitochondrial diseases.
  17. Adipocyte-specific inactivation of NAMPT, a key NAD+ biosynthetic enzyme, causes a metabolically-unhealthy lean phenotype in female mice during aging.
  18. Development and characterization of phospho-ubiquitin antibodies to monitor PINK1-PRKN signaling in cells and tissue.
  19. ANT2 functions as a translocon for mitochondrial cross-membrane translocation of RNAs.
  20. Measuring the forces that shape early human embryos.
  21. Nanobodies as novel tools to monitor the mitochondrial fission factor Drp1.
  22. Structural basis of substrate recognition and allosteric activation of the proapoptotic mitochondrial HtrA2 protease.
  23. Exploring mitochondrial heteroplasmy in neonates: implications for growth patterns and overweight in the first years of life.
  24. Transport activity regulates mitochondrial bioenergetics and biogenesis in renal tubules.
  25. GSK-3α-BNIP3 axis promotes mitophagy in human cardiomyocytes under hypoxia.
  1. Sci Adv. 2024 May 31. 10(22): eadn2050
    CLUH maintains functional mitochondria and translation in motoneuronal axons and prevents peripheral neuropathy.
    Marta Zaninello, Tim Schlegel, Hendrik Nolte, Mujeeb Pirzada, Elisa Savino, Esther Barth, Ines Klein, Hauke Wüstenberg, Tesmin Uddin, Lisa Wolff, Brunhilde Wirth, Helmar C Lehmann, Jean-Michel Cioni, Thomas Langer, Elena I Rugarli.
      Transporting and translating mRNAs in axons is crucial for neuronal viability. Local synthesis of nuclear-encoded mitochondrial proteins protects long-lived axonal mitochondria from damage; however, the regulatory factors involved are largely unknown. We show that CLUH, which binds mRNAs encoding mitochondrial proteins, prevents peripheral neuropathy and motor deficits in the mouse. CLUH is enriched in the growth cone of developing spinal motoneurons and is required for their growth. The lack of CLUH affects the abundance of target mRNAs and the corresponding mitochondrial proteins more prominently in axons, leading to ATP deficits in the growth cone. CLUH interacts with ribosomal subunits, translation initiation, and ribosome recycling components and preserves axonal translation. Overexpression of the ribosome recycling factor ABCE1 rescues the mRNA and translation defects, as well as the growth cone size, in CLUH-deficient motoneurons. Thus, we demonstrate a role for CLUH in mitochondrial quality control and translational regulation in axons, which is essential for their development and long-term integrity and function.
    DOI:  https://doi.org/10.1126/sciadv.adn2050
  2. Nat Nanotechnol. 2024 May 27.
    Oral mitochondrial transplantation using nanomotors to treat ischaemic heart disease.
    Ziyu Wu, Lin Chen, Wenyan Guo, Jun Wang, Haiya Ni, Jianing Liu, Wentao Jiang, Jian Shen, Chun Mao, Min Zhou, Mimi Wan.
      Mitochondrial transplantation is an important therapeutic strategy for restoring energy supply in patients with ischaemic heart disease (IHD); however, it is limited by the invasiveness of the transplantation method and loss of mitochondrial activity. Here we report successful mitochondrial transplantation by oral administration for IHD therapy. A nitric-oxide-releasing nanomotor is modified on the mitochondria surface to obtain nanomotorized mitochondria with chemotactic targeting ability towards damaged heart tissue due to nanomotor action. The nanomotorized mitochondria are packaged in enteric capsules to protect them from gastric acid erosion. After oral delivery the mitochondria are released in the intestine, where they are quickly absorbed by intestinal cells and secreted into the bloodstream, allowing delivery to the damaged heart tissue. The regulation of disease microenvironment by the nanomotorized mitochondria can not only achieve rapid uptake and high retention of mitochondria by damaged cardiomyocytes but also maintains high activity of the transplanted mitochondria. Furthermore, results from animal models of IHD indicate that the accumulated nanomotorized mitochondria in the damaged heart tissue can regulate cardiac metabolism at the transcriptional level, thus preventing IHD progression. This strategy has the potential to change the therapeutic strategy used to treat IHD.
    DOI:  https://doi.org/10.1038/s41565-024-01681-7
  3. Biochem J. 2024 Jun 05. 481(11): 683-715
    Mechanisms and pathologies of human mitochondrial DNA replication and deletion formation.
    Tiago M Bernardino Gomes, Amy E Vincent, Katja E Menger, James B Stewart, Thomas J Nicholls.
      Human mitochondria possess a multi-copy circular genome, mitochondrial DNA (mtDNA), that is essential for cellular energy metabolism. The number of copies of mtDNA per cell, and their integrity, are maintained by nuclear-encoded mtDNA replication and repair machineries. Aberrant mtDNA replication and mtDNA breakage are believed to cause deletions within mtDNA. The genomic location and breakpoint sequences of these deletions show similar patterns across various inherited and acquired diseases, and are also observed during normal ageing, suggesting a common mechanism of deletion formation. However, an ongoing debate over the mechanism by which mtDNA replicates has made it difficult to develop clear and testable models for how mtDNA rearrangements arise and propagate at a molecular and cellular level. These deletions may impair energy metabolism if present in a high proportion of the mtDNA copies within the cell, and can be seen in primary mitochondrial diseases, either in sporadic cases or caused by autosomal variants in nuclear-encoded mtDNA maintenance genes. These mitochondrial diseases have diverse genetic causes and multiple modes of inheritance, and show notoriously broad clinical heterogeneity with complex tissue specificities, which further makes establishing genotype-phenotype relationships challenging. In this review, we aim to cover our current understanding of how the human mitochondrial genome is replicated, the mechanisms by which mtDNA replication and repair can lead to mtDNA instability in the form of large-scale rearrangements, how rearranged mtDNAs subsequently accumulate within cells, and the pathological consequences when this occurs.
    Keywords:  DNA damage; DNA replication and recombination; mitochondrial dysfunction; mtDNA
    DOI:  https://doi.org/10.1042/BCJ20230262
  4. Cell Rep. 2024 May 29. pii: S2211-1247(24)00622-3. [Epub ahead of print]43(6): 114294
    TBK1 is ubiquitinated by TRIM5α to assemble mitophagy machinery.
    Bhaskar Saha, Hallvard Olsvik, Geneva L Williams, Seeun Oh, Gry Evjen, Eva Sjøttem, Michael A Mandell.
      Ubiquitination of mitochondrial proteins provides a basis for the downstream recruitment of mitophagy machinery, yet whether ubiquitination of the machinery itself contributes to mitophagy is unknown. Here, we show that K63-linked polyubiquitination of the key mitophagy regulator TBK1 is essential for its mitophagy functions. This modification is catalyzed by the ubiquitin ligase TRIM5α and is required for TBK1 to interact with and activate a set of ubiquitin-binding autophagy adaptors including NDP52, p62/SQSTM1, and NBR1. Autophagy adaptors, along with TRIM27, enable TRIM5α to engage with TBK1 following mitochondrial damage. TRIM5α's ubiquitin ligase activity is required for the accumulation of active TBK1 on damaged mitochondria in Parkin-dependent and Parkin-independent mitophagy pathways. Our data support a model in which TRIM5α provides a mitochondria-localized, ubiquitin-based, self-amplifying assembly platform for TBK1 and mitophagy adaptors that is ultimately necessary for the recruitment of the core autophagy machinery.
    Keywords:  CP: Cell biology; NBR1; NDP52; Optineurin; TAX1BP1; TBK1; TRIM27/RFP; autophagy; p62; tripartite motif; ubiquitin ligase
    DOI:  https://doi.org/10.1016/j.celrep.2024.114294
  5. Nat Cell Biol. 2024 May 29.
    Cells dispose of cytoplasmic mitochondrial DNA by nucleoid-phagy.
      
    DOI:  https://doi.org/10.1038/s41556-024-01421-y
  6. Cell Death Dis. 2024 May 31. 15(5): 382
    Targeting the autophagy-NAD axis protects against cell death in Niemann-Pick type C1 disease models.
    Tetsushi Kataura, Lucia Sedlackova, Congxin Sun, Gamze Kocak, Niall Wilson, Peter Banks, Faisal Hayat, Sergey Trushin, Eugenia Trushina, Oliver D K Maddocks, John E Oblong, Satomi Miwa, Masaya Imoto, Shinji Saiki, Daniel Erskine, Marie E Migaud, Sovan Sarkar, Viktor I Korolchuk.
      Impairment of autophagy leads to an accumulation of misfolded proteins and damaged organelles and has been implicated in plethora of human diseases. Loss of autophagy in actively respiring cells has also been shown to trigger metabolic collapse mediated by the depletion of nicotinamide adenine dinucleotide (NAD) pools, resulting in cell death. Here we found that the deficit in the autophagy-NAD axis underpins the loss of viability in cell models of a neurodegenerative lysosomal storage disorder, Niemann-Pick type C1 (NPC1) disease. Defective autophagic flux in NPC1 cells resulted in mitochondrial dysfunction due to impairment of mitophagy, leading to the depletion of both the reduced and oxidised forms of NAD as identified via metabolic profiling. Consequently, exhaustion of the NAD pools triggered mitochondrial depolarisation and apoptotic cell death. Our chemical screening identified two FDA-approved drugs, celecoxib and memantine, as autophagy activators which effectively restored autophagic flux, NAD levels, and cell viability of NPC1 cells. Of biomedical relevance, either pharmacological rescue of the autophagy deficiency or NAD precursor supplementation restored NAD levels and improved the viability of NPC1 patient fibroblasts and induced pluripotent stem cell (iPSC)-derived cortical neurons. Together, our findings identify the autophagy-NAD axis as a mechanism of cell death and a target for therapeutic interventions in NPC1 disease, with a potential relevance to other neurodegenerative disorders.
    DOI:  https://doi.org/10.1038/s41419-024-06770-y
  7. Cell Death Dis. 2024 May 25. 15(5): 361
    Mitochondrial iron deficiency triggers cytosolic iron overload in PKAN hiPS-derived astrocytes.
    Paolo Santambrogio, Anna Cozzi, Chiara Balestrucci, Maddalena Ripamonti, Valeria Berno, Eugenia Cammarota, Andrea Stefano Moro, Sonia Levi.
      Disease models of neurodegeneration with brain iron accumulation (NBIA) offer the possibility to explore the relationship between iron dyshomeostasis and neurodegeneration. We analyzed hiPS-derived astrocytes from PANK2-associated neurodegeneration (PKAN), an NBIA disease characterized by progressive neurodegeneration and high iron accumulation in the globus pallidus. Previous data indicated that PKAN astrocytes exhibit alterations in iron metabolism, general impairment of constitutive endosomal trafficking, mitochondrial dysfunction and acquired neurotoxic features. Here, we performed a more in-depth analysis of the interactions between endocytic vesicles and mitochondria via superresolution microscopy experiments. A significantly lower number of transferrin-enriched vesicles were in contact with mitochondria in PKAN cells than in control cells, confirming the impaired intracellular fate of cargo endosomes. The investigation of cytosolic and mitochondrial iron parameters indicated that mitochondrial iron availability was substantially lower in PKAN cells compared to that in the controls. In addition, PKAN astrocytes exhibited defects in tubulin acetylation/phosphorylation, which might be responsible for unregulated vesicular dynamics and inappropriate iron delivery to mitochondria. Thus, the impairment of iron incorporation into these organelles seems to be the cause of cell iron delocalization, resulting in cytosolic iron overload and mitochondrial iron deficiency, triggering mitochondrial dysfunction. Overall, the data elucidate the mechanism of iron accumulation in CoA deficiency, highlighting the importance of mitochondrial iron deficiency in the pathogenesis of disease.
    DOI:  https://doi.org/10.1038/s41419-024-06757-9
  8. Mil Med Res. 2024 May 29. 11(1): 32
    Mitochondrial quality control in human health and disease.
    Bo-Hao Liu, Chen-Zhen Xu, Yi Liu, Zi-Long Lu, Ting-Lv Fu, Guo-Rui Li, Yu Deng, Guo-Qing Luo, Song Ding, Ning Li, Qing Geng.
      Mitochondria, the most crucial energy-generating organelles in eukaryotic cells, play a pivotal role in regulating energy metabolism. However, their significance extends beyond this, as they are also indispensable in vital life processes such as cell proliferation, differentiation, immune responses, and redox balance. In response to various physiological signals or external stimuli, a sophisticated mitochondrial quality control (MQC) mechanism has evolved, encompassing key processes like mitochondrial biogenesis, mitochondrial dynamics, and mitophagy, which have garnered increasing attention from researchers to unveil their specific molecular mechanisms. In this review, we present a comprehensive summary of the primary mechanisms and functions of key regulators involved in major components of MQC. Furthermore, the critical physiological functions regulated by MQC and its diverse roles in the progression of various systemic diseases have been described in detail. We also discuss agonists or antagonists targeting MQC, aiming to explore potential therapeutic and research prospects by enhancing MQC to stabilize mitochondrial function.
    Keywords:  Cancer; Cardiovascular disease; Digestive system disease; Kidney disease; Metabolic disease; Metabolism; Mitochondrial quality control; Nervous disease; Programmed cell death; Pulmonary disease
    DOI:  https://doi.org/10.1186/s40779-024-00536-5
  9. J Mol Biol. 2024 May 29. pii: S0022-2836(24)00226-2. [Epub ahead of print] 168631
    FIP200 phosphorylation regulates late steps in mitophagy.
    Christopher Eickhorst, Riccardo Babic, Jorrell Rush-Kittle, Leon Lucya, Fatimah Lami Imam, Pablo Sánchez-Martín, David M Hollenstein, Jonas Michaelis, Christian Münch, Chris Meisinger, Dea Slade, Laura Gámez-Díaz, Claudine Kraft.
      Mitophagy is a specific type of autophagy responsible for the selective elimination of dysfunctional or superfluous mitochondria, ensuring the maintenance of mitochondrial quality control. The initiation of mitophagy is coordinated by the ULK1 kinase complex, which engages mitophagy receptors via its FIP200 subunit. Whether FIP200 performs additional functions in the subsequent later phases of mitophagy beyond this initial step and how its regulation occurs, remains unclear. Our findings reveal that multiple phosphorylation events on FIP200 differentially control the early and late stages of mitophagy. Furthermore, these phosphorylation events influence FIP200's interaction with ATG16L1. In summary, our results highlight the necessity for precise and dynamic regulation of FIP200, underscoring its importance in the progression of mitophagy.
    Keywords:  ATG16L1; Atg1/ULK1 kinase complex; FIP200; autophagy; mitophagy
    DOI:  https://doi.org/10.1016/j.jmb.2024.168631
  10. Front Cell Dev Biol. 2024 ;12 1346778
    Spatial detection of mitochondrial DNA and RNA in tissues.
    Michelle Giarmarco, Jordan Seto, Daniel Brock, Susan Brockerhoff.
       Background: Mitochondrial health has gained attention in a number of diseases, both as an indicator of disease state and as a potential therapeutic target. The quality and amount of mitochondrial DNA (mtDNA) and RNA (mtRNA) can be important indicators of mitochondrial and cell health, but are difficult to measure in complex tissues.
    Methods: mtDNA and mtRNA in zebrafish retina samples were fluorescently labeled using RNAscope™ in situ hybridization, then mitochondria were stained using immunohistochemistry. Pretreatment with RNase was used for validation. Confocal images were collected and analyzed, and relative amounts of mtDNA and mtRNA were reported. Findings regarding mtDNA were confirmed using qPCR.
    Results: Signals from probes detecting mtDNA and mtRNA were localized to mitochondria, and were differentially sensitive to RNase. This labeling strategy allows for quantification of relative mtDNA and mtRNA levels in individual cells. As a demonstration of the method in a complex tissue, single photoreceptors in zebrafish retina were analyzed for mtDNA and mtRNA content. An increase in mtRNA but not mtDNA coincides with proliferation of mitochondria at night in cones. A similar trend was measured in rods.
    Discussion: Mitochondrial gene expression is an important component of cell adaptations to disease, stress, or aging. This method enables the study of mtDNA and mtRNA in single cells of an intact, complex tissue. The protocol presented here uses commercially-available tools, and is adaptable to a range of species and tissue types.
    Keywords:  RNAscope; mitochondria; mtDNA; mtRNA; multiplex imaging; photoreceptor; spatial biology; zebrafish
    DOI:  https://doi.org/10.3389/fcell.2024.1346778
  11. Autophagy. 2024 May 31.
    Atg44/Mdi1/mitofissin facilitates Dnm1-mediated mitochondrial fission.
    Kentaro Furukawa, Manabu Hayatsu, Kentaro Okuyama, Tomoyuki Fukuda, Shun-Ichi Yamashita, Keiichi Inoue, Shinsuke Shibata, Tomotake Kanki.
      Mitochondria undergo fission and fusion, and their coordinated balance is crucial for maintaining mitochondrial homeostasis. In yeast, the dynamin-related protein Dnm1 is a mitochondrial fission factor acting from outside the mitochondria. We recently reported the mitochondrial intermembrane space protein Atg44/mitofissin/Mdi1/Mco8 as a novel fission factor, but the relationship between Atg44 and Dnm1 remains elusive. Here, we show that Atg44 is required to complete Dnm1-mediated mitochondrial fission under homeostatic conditions. Atg44-deficient cells often exhibit enlarged mitochondria with accumulated Dnm1 and rosary-like mitochondria with Dnm1 foci at constriction sites. These mitochondrial constriction sites retain the continuity of both the outer and inner membranes within an extremely confined space, indicating that Dnm1 is unable to complete mitochondrial fission without Atg44. Moreover, accumulated Atg44 proteins are observed at mitochondrial constriction sites. These findings suggest that Atg44 and Dnm1 cooperatively execute mitochondrial fission from inside and outside the mitochondria, respectively.
    Keywords:  Atg44; Dnm1; mitochondrial fission; mitofissin; mitophagy; yeast
    DOI:  https://doi.org/10.1080/15548627.2024.2360345
  12. NPJ Metab Health Dis. 2024 ;2(1): 6
    Multifaceted mitochondria in innate immunity.
    Eloïse Marques, Robbin Kramer, Dylan G Ryan.
      The ability of mitochondria to transform the energy we obtain from food into cell phosphorylation potential has long been appreciated. However, recent decades have seen an evolution in our understanding of mitochondria, highlighting their significance as key signal-transducing organelles with essential roles in immunity that extend beyond their bioenergetic function. Importantly, mitochondria retain bacterial motifs as a remnant of their endosymbiotic origin that are recognised by innate immune cells to trigger inflammation and participate in anti-microbial defence. This review aims to explore how mitochondrial physiology, spanning from oxidative phosphorylation (OxPhos) to signalling of mitochondrial nucleic acids, metabolites, and lipids, influences the effector functions of phagocytes. These myriad effector functions include macrophage polarisation, efferocytosis, anti-bactericidal activity, antigen presentation, immune signalling, and cytokine regulation. Strict regulation of these processes is critical for organismal homeostasis that when disrupted may cause injury or contribute to disease. Thus, the expanding body of literature, which continues to highlight the central role of mitochondria in the innate immune system, may provide insights for the development of the next generation of therapies for inflammatory diseases.
    Keywords:  Energy metabolism; Mitochondria
    DOI:  https://doi.org/10.1038/s44324-024-00008-3
  13. Front Cell Dev Biol. 2024 ;12 1403463
    Mitochondrial defects in sporadic inclusion body myositis-causes and consequences.
    Elsie Chit Yu Iu, Ho So, Chi Bun Chan.
      Sporadic inclusion body myositis (sIBM) is a distinct subcategory of Idiopathic Inflammatory Myopathies (IIM), characterized by unique pathological features such as muscle inflammation, rimmed vacuoles, and protein aggregation within the myofibers. Although hyperactivation of the immune system is widely believed as the primary cause of IIM, it is debated whether non-immune tissue dysfunction might contribute to the disease's onset as patients with sIBM are refractory to conventional immunosuppressant treatment. Moreover, the findings that mitochondrial dysfunction can elicit non-apoptotic programmed cell death and the subsequent immune response further support this hypothesis. Notably, abnormal mitochondrial structure and activities are more prominent in the muscle of sIBM than in other types of IIM, suggesting the presence of defective mitochondria might represent an overlooked contributor to the disease onset. The large-scale mitochondrial DNA deletion, aberrant protein aggregation, and slowed organelle turnover have provided mechanistic insights into the genesis of impaired mitochondria in sIBM. This article reviews the disease hallmarks of sIBM, the plausible contributors of mitochondrial damage in the sIBM muscle, and the immunological responses associated with mitochondrial perturbations. Additionally, the potential application of mitochondrial-targeted chemicals as a new treatment strategy to sIBM is explored and discussed.
    Keywords:  mitochondria; muscle; myositis; necroptosis; pathogenesis; pyroptosis
    DOI:  https://doi.org/10.3389/fcell.2024.1403463
  14. Mitochondrion. 2024 May 24. pii: S1567-7249(24)00063-1. [Epub ahead of print]78 101905
    ACAD9 treatment with bezafibrate and nicotinamide riboside temporarily stabilizes cardiomyopathy and lactic acidosis.
    Johan L K Van Hove, Marisa W Friederich, Daniella H Hock, David A Stroud, Nikeisha J Caruana, Uwe Christians, Björn Schniedewind, Cole R Michel, Richard Reisdorph, Edwin D J Lopez Gonzalez, Charles Brenner, Tonia E Donovan, Jessica C Lee, Kathryn C Chatfield, Austin A Larson, Peter R Baker, Shawn E McCandless, Meghan F Moore Burk.
      Pathogenic ACAD9 variants cause complex I deficiency. Patients presenting in infancy unresponsive to riboflavin have high mortality. A six-month-old infant presented with riboflavin unresponsive lactic acidosis and life-threatening cardiomyopathy. Treatment with high dose bezafibrate and nicotinamide riboside resulted in marked clinical improvement including reduced lactate and NT-pro-brain type natriuretic peptide levels, with stabilized echocardiographic measures. After a long stable period, the child succumbed from cardiac failure with infection at 10.5 months. Therapy was well tolerated. Peak bezafibrate levels exceeded its EC50. The clinical improvement with this treatment illustrates its potential, but weak PPAR agonist activity of bezafibrate limited its efficacy.
    Keywords:  ACAD9 disorder; Bezafibrate; Cardiomyopathy; Mitochondrial disease; Nicotinamide riboside; Treatment; complex I deficiency
    DOI:  https://doi.org/10.1016/j.mito.2024.101905
  15. Mol Ther. 2024 May 24. pii: S1525-0016(24)00332-0. [Epub ahead of print]
    Neuroglobin overexpression in cerebellar neurons of Harlequin mice improves mitochondrial homeostasis and reduces ataxic behavior.
    Hélène Cwerman-Thibault, Vassilissa Malko-Baverel, Gwendoline Le Guilloux, Edward Ratcliffe, Djmila Mouri, Isabel Torres-Cuevas, Ivan Millán, Bruno Saubaméa, Virginie Mignon, Odile Boespflug-Tanguy, Pierre Gressens, Marisol Corral-Debrinski.
      Neuroglobin, a member of the globin superfamily, is abundant in the brain, retina and cerebellum of mammals and localizes to mitochondria. The protein exhibits neuroprotective capacities by participating to electron transfer, oxygen supply and protecting against oxidative stress. Our objective is to determine whether Neuroglobin overexpression can be used to treat neurological disorders. We chose Harlequin mice, which harbor a retroviral insertion in the first intron of the Apoptosis Inducing Factor gene resulting in the depletion of the corresponding protein essential for mitochondrial biogenesis. Consequently, Harlequin mice display degeneration of the cerebellum and suffer from progressive blindness and ataxia. Cerebellar ataxia begins in Harlequin mice at the age of four months and is characterized by neuronal cell disappearance, bioenergetics failure, motor and cognitive impairments which aggravated with aging. Mice aged two months received Adeno-Associated Viral vectors harboring the coding sequence of Neuroglobin or Apoptosis-inducing factor in both cerebellar hemispheres. Six months later, Harlequin mice exhibited substantial improvements in motor and cognitive skills; probably linked to the preservation of respiratory chain function, Purkinje cell numbers and connectivity. Thus, without sharing functional properties with Apoptosis-inducing factor, neuroglobin was efficient to reduce ataxia in Harlequin mice.
    DOI:  https://doi.org/10.1016/j.ymthe.2024.05.030
  16. Trends Endocrinol Metab. 2024 May 27. pii: S1043-2760(24)00119-X. [Epub ahead of print]
    ISR pathway contribution to tissue specificity of mitochondrial diseases.
    Ana Vela-Sebastián, Pilar Bayona-Bafaluy, David Pacheu-Grau.
      Mitochondrial genetic defects caused by whole-body mutations typically affect different tissues in different ways. Elucidating the molecular determinants that cause certain cell types to be primarily affected has become a critical research target within the field. We propose a differential activation of the integrated stress response as a potential contributor to this tissue specificity.
    DOI:  https://doi.org/10.1016/j.tem.2024.05.001
  17. Am J Physiol Endocrinol Metab. 2024 May 29.
    Adipocyte-specific inactivation of NAMPT, a key NAD+ biosynthetic enzyme, causes a metabolically-unhealthy lean phenotype in female mice during aging.
    Nathan Qi, Michael P Franczyk, Shintaro Yamaguchi, Daiki Kojima, Kaori Hayashi, Akiko Satoh, Noboru Ogiso, Takeshi Kanda, Yo Sasaki, Brian N Finck, Brian J DeBosch, Jun Yoshino.
      Nicotinamide adenine dinucleotide (NAD+) is a universal coenzyme regulating cellular energy metabolism in many cell types. Recent studies have demonstrated the close relationships between defective NAD+ metabolism and aging and age-associated metabolic diseases. The major purpose of the present study was to test the hypothesis that NAD+ biosynthesis, mediated by a rate-limiting NAD+ biosynthetic enzyme, nicotinamide phosphoribosyltransferase (NAMPT), is essential for maintaining normal adipose tissue function and whole-body metabolic health during the aging process. To this end, we provided in-depth and comprehensive metabolic assessments for female adipocyte-specific Nampt knockout (ANKO) mice during aging. We first evaluated body fat mass in young (≤ 4-month-old), middle aged (10 to 14-month-old), and old (≥ 18-month-old) mice. Intriguingly, adipocyte-specific Nampt deletion protected against age-induced obesity without changing energy balance. However, data obtained from the hyperinsulinemic euglycemic clamp procedure demonstrated that, despite the lean phenotype, old ANKO mice had severe insulin resistance in skeletal muscle, heart, and white adipose tissue (WAT). Old ANKO mice also exhibited hyperinsulinemia and hypoadiponectinemia. Mechanistically, loss of Nampt caused marked decreases in WAT gene expression of lipogenic targets of peroxisome proliferator-activated receptor gamma (PPARγ) in an age-dependent manner. In addition, administration of a PPARγ agonist rosiglitazone restored fat mass and improved metabolic abnormalities in old ANKO mice. In conclusion, these findings highlight the importance of the NAMPT-NAD+-PPARγ axis in maintaining functional integrity and quantity of adipose tissue, and whole-body metabolic function in female mice during aging.
    Keywords:  Adipose tissue; NAD; PPARγ; aging; insulin resistance
    DOI:  https://doi.org/10.1152/ajpendo.00313.2023
  18. Autophagy. 2024 May 27. 1-16
    Development and characterization of phospho-ubiquitin antibodies to monitor PINK1-PRKN signaling in cells and tissue.
    Jens O Watzlawik, Xu Hou, Tyrique Richardson, Szymon L Lewicki, Joanna Siuda, Zbigniew K Wszolek, Casey N Cook, Leonard Petrucelli, Michael DeTure, Dennis W Dickson, Odetta Antico, Miratul M K Muqit, Jordan B Fishman, Karima Pirani, Ravindran Kumaran, Nicole K Polinski, Fabienne C Fiesel, Wolfdieter Springer.
      The selective removal of dysfunctional mitochondria, a process termed mitophagy, is critical for cellular health and impairments have been linked to aging, Parkinson disease, and other neurodegenerative conditions. A central mitophagy pathway is orchestrated by the ubiquitin (Ub) kinase PINK1 together with the E3 Ub ligase PRKN/Parkin. The decoration of damaged mitochondrial domains with phosphorylated Ub (p-S65-Ub) mediates their elimination though the autophagy system. As such p-S65-Ub has emerged as a highly specific and quantitative marker of mitochondrial damage with significant disease relevance. Existing p-S65-Ub antibodies have been successfully employed as research tools in a range of applications including western blot, immunocytochemistry, immunohistochemistry, and enzyme-linked immunosorbent assay. However, physiological levels of p-S65-Ub in the absence of exogenous stress are very low, therefore difficult to detect and require reliable and ultrasensitive methods. Here we generated and characterized a collection of novel recombinant, rabbit monoclonal p-S65-Ub antibodies with high specificity and affinity in certain applications that allow the field to better understand the molecular mechanisms and disease relevance of PINK1-PRKN signaling. These antibodies may also serve as novel diagnostic or prognostic tools to monitor mitochondrial damage in various clinical and pathological specimens.Abbreviations: AD: Alzheimer disease; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; ELISA: enzyme-linked immunosorbent assay; HEK293E cell: human embryonic kidney E cell; ICC: immunocytochemistry; IHC: immunohistochemistry: KO: knockout; LoB: limit of blank; LoD: limit of detection; LoQ: limit of quantification; MEF: mouse embryonic fibroblast; MSD: Meso Scale Discovery; n.s.: non-significant; nonTg: non-transgenic; PBMC: peripheral blood mononuclear cell; PD: Parkinson disease; p-S65-PRKN: phosphorylated PRKN at serine 65; p-S65-Ub: phosphorylated Ub at serine 65; Ub: ubiquitin; WT: wild-type.
    Keywords:  Autophagy; PINK1; Parkinson disease; mitochondria; mitophagy; ubiquitin
    DOI:  https://doi.org/10.1080/15548627.2024.2356490
  19. Cell Res. 2024 May 29.
    ANT2 functions as a translocon for mitochondrial cross-membrane translocation of RNAs.
    Pengcheng Wang, Lixiao Zhang, Siyi Chen, Renjian Li, Peipei Liu, Xiang Li, Hongdi Luo, Yujia Huo, Zhirong Zhang, Yiqi Cai, Xu Liu, Jinliang Huang, Guangkeng Zhou, Zhe Sun, Shanwei Ding, Jiahao Shi, Zizhuo Zhou, Ruoxi Yuan, Liang Liu, Sipeng Wu, Geng Wang.
      Bidirectional transcription of mammalian mitochondrial DNA generates overlapping transcripts that are capable of forming double-stranded RNA (dsRNA) structures. Release of mitochondrial dsRNA into the cytosol activates the dsRNA-sensing immune signaling, which is a defense mechanism against microbial and viral attack and possibly cancer, but could cause autoimmune diseases when unchecked. A better understanding of the process is vital in therapeutic application of this defense mechanism and treatment of cognate human diseases. In addition to exporting dsRNAs, mitochondria also export and import a variety of non-coding RNAs. However, little is known about how these RNAs are transported across mitochondrial membranes. Here we provide direct evidence showing that adenine nucleotide translocase-2 (ANT2) functions as a mammalian RNA translocon in the mitochondrial inner membrane, independent of its ADP/ATP translocase activity. We also show that mitochondrial dsRNA efflux through ANT2 triggers innate immunity. Inhibiting this process alleviates inflammation in vivo, providing a potential therapeutic approach for treating autoimmune diseases.
    DOI:  https://doi.org/10.1038/s41422-024-00978-5
  20. Nature. 2024 May 30.
    Measuring the forces that shape early human embryos.
      
    Keywords:  Biological techniques; Developmental biology
    DOI:  https://doi.org/10.1038/d41586-024-01556-w
  21. Life Sci Alliance. 2024 Aug;pii: e202402608. [Epub ahead of print]7(8):
    Nanobodies as novel tools to monitor the mitochondrial fission factor Drp1.
    Theresa Froehlich, Andreas Jenner, Claudia Cavarischia-Rega, Funmilayo O Fagbadebo, Yannic Lurz, Desiree I Frecot, Philipp D Kaiser, Stefan Nueske, Armin M Scholz, Erik Schäffer, Ana J Garcia-Saez, Boris Macek, Ulrich Rothbauer.
      In cells, mitochondria undergo constant fusion and fission. An essential factor for fission is the mammalian dynamin-related protein 1 (Drp1). Dysregulation of Drp1 is associated with neurodegenerative diseases including Parkinson's, cardiovascular diseases and cancer, making Drp1 a pivotal biomarker for monitoring mitochondrial status and potential pathophysiological conditions. Here, we developed nanobodies (Nbs) as versatile binding molecules for proteomics, advanced microscopy and live cell imaging of Drp1. To specifically enrich endogenous Drp1 with interacting proteins for proteomics, we functionalized high-affinity Nbs into advanced capture matrices. Furthermore, we detected Drp1 by bivalent Nbs combined with site-directed fluorophore labelling in super-resolution STORM microscopy. For real-time imaging of Drp1, we intracellularly expressed fluorescently labelled Nbs, so-called chromobodies (Cbs). To improve the signal-to-noise ratio, we further converted Cbs into a "turnover-accelerated" format. With these imaging probes, we visualized the dynamics of endogenous Drp1 upon compound-induced mitochondrial fission in living cells. Considering the wide range of research applications, the presented Nb toolset will open up new possibilities for advanced functional studies of Drp1 in disease-relevant models.
    DOI:  https://doi.org/10.26508/lsa.202402608
  22. Nat Commun. 2024 May 30. 15(1): 4592
    Structural basis of substrate recognition and allosteric activation of the proapoptotic mitochondrial HtrA2 protease.
    Emelie E Aspholm, Jens Lidman, Björn M Burmann.
      The mitochondrial serine protease HtrA2 is a human homolog of the Escherichia coli Deg-proteins exhibiting chaperone and proteolytic roles. HtrA2 is involved in both apoptotic regulation via its ability to degrade inhibitor-of-apoptosis proteins (IAPs), as well as in cellular maintenance as part of the cellular protein quality control machinery, by preventing the possible toxic accumulation of aggregated proteins. In this study, we use advanced solution NMR spectroscopy methods combined with biophysical characterization and biochemical assays to elucidate the crucial role of the substrate recognizing PDZ domain. This domain regulates the protease activity of HtrA2 by triggering an intricate allosteric network involving the regulatory loops of the protease domain. We further show that divalent metal ions can both positively and negatively modulate the activity of HtrA2, leading to a refined model of HtrA2 regulation within the apoptotic pathway.
    DOI:  https://doi.org/10.1038/s41467-024-48997-5
  23. Int J Obes (Lond). 2024 May 27.
    Exploring mitochondrial heteroplasmy in neonates: implications for growth patterns and overweight in the first years of life.
    Charlotte Cosemans, Rossella Alfano, Hanne Sleurs, Dries S Martens, Tim S Nawrot, Michelle Plusquin.
       BACKGROUND: Mitochondrial heteroplasmy reflects genetic diversity within individuals due to the presence of varying mitochondrial DNA (mtDNA) sequences, possibly affecting mitochondrial function and energy production in cells. Rapid growth during early childhood is a critical development with long-term implications for health and well-being. In this study, we investigated if cord blood mtDNA heteroplasmy is associated with rapid growth at 6 and 12 months and overweight in childhood at 4-6 years.
    METHODS: This study included 200 mother-child pairs of the ENVIRONAGE birth cohort. Whole mitochondrial genome sequencing was performed to determine mtDNA heteroplasmy levels (in variant allele frequency; VAF) in cord blood. Rapid growth was defined for each child as the difference between WHO-SD scores of predicted weight at either 6 or 12 months and birth weight. Logistic regression models were used to determine the association of mitochondrial heteroplasmy with rapid growth and childhood overweight. Determinants of relevant cord blood mitochondrial heteroplasmies were identified using multiple linear regression models.
    RESULTS: One % increase in VAF of cord blood MT-D-Loop16362T > C heteroplasmy was associated with rapid growth at 6 months (OR = 1.03; 95% CI: 1.01-1.05; p = 0.001) and 12 months (OR = 1.02; 95% CI: 1.00-1.03; p = 0.02). Furthermore, this variant was associated with childhood overweight at 4-6 years (OR = 1.01; 95% CI 1.00-1.02; p = 0.05). Additionally, rapid growth at 6 months (OR = 3.00; 95% CI: 1.49-6.14; p = 0.002) and 12 months (OR = 4.05; 95% CI: 2.06-8.49; p < 0.001) was also associated with childhood overweight at 4-6 years. Furthermore, we identified maternal age, pre-pregnancy BMI, maternal education, parity, and gestational age as determinants of cord blood MT-D-Loop16362T > C heteroplasmy.
    CONCLUSIONS: Our findings, based on mitochondrial DNA genotyping, offer insights into the molecular machinery leading to rapid growth in early life, potentially explaining a working mechanism of the development toward childhood overweight.
    DOI:  https://doi.org/10.1038/s41366-024-01537-z
  24. FASEB J. 2024 May 31. 38(10): e23703
    Transport activity regulates mitochondrial bioenergetics and biogenesis in renal tubules.
    Chih-Jen Cheng, Jonathan M Nizar, Dao-Fu Dai, Chou-Long Huang.
      Renal tubules are featured with copious mitochondria and robust transport activity. Mutations in mitochondrial genes cause congenital renal tubulopathies, and changes in transport activity affect mitochondrial morphology, suggesting mitochondrial function and transport activity are tightly coupled. Current methods of using bulk kidney tissues or cultured cells to study mitochondrial bioenergetics are limited. Here, we optimized an extracellular flux analysis (EFA) to study mitochondrial respiration and energy metabolism using microdissected mouse renal tubule segments. EFA detects mitochondrial respiration and glycolysis by measuring oxygen consumption and extracellular acidification rates, respectively. We show that both measurements positively correlate with sample sizes of a few centimeter-length renal tubules. The thick ascending limbs (TALs) and distal convoluted tubules (DCTs) critically utilize glucose/pyruvate as energy substrates, whereas proximal tubules (PTs) are significantly much less so. Acute inhibition of TALs' transport activity by ouabain treatment reduces basal and ATP-linked mitochondrial respiration. Chronic inhibition of transport activity by 2-week furosemide treatment or deletion of with-no-lysine kinase 4 (Wnk4) decreases maximal mitochondrial capacity. In addition, chronic inhibition downregulates mitochondrial DNA mass and mitochondrial length/density in TALs and DCTs. Conversely, gain-of-function Wnk4 mutation increases maximal mitochondrial capacity and mitochondrial length/density without increasing mitochondrial DNA mass. In conclusion, EFA is a sensitive and reliable method to investigate mitochondrial functions in isolated renal tubules. Transport activity tightly regulates mitochondrial bioenergetics and biogenesis to meet the energy demand in renal tubules. The system allows future investigation into whether and how mitochondria contribute to tubular remodeling adapted to changes in transport activity.
    Keywords:  distal convoluted tubule; extracellular flux analysis; glycolysis; mitochondrial respiration; thick ascending limb; transport activity
    DOI:  https://doi.org/10.1096/fj.202400358RR
  25. Free Radic Biol Med. 2024 May 28. pii: S0891-5849(24)00498-2. [Epub ahead of print]221 235-244
    GSK-3α-BNIP3 axis promotes mitophagy in human cardiomyocytes under hypoxia.
    Hezlin Marzook, Anamika Gupta, Manju N Jayakumar, Mohamed A Saleh, Dhanendra Tomar, Rizwan Qaisar, Firdos Ahmad.
      Dysregulated autophagy/mitophagy is one of the major causes of cardiac injury in ischemic conditions. Glycogen synthase kinase-3alpha (GSK-3α) has been shown to play a crucial role in the pathophysiology of cardiac diseases. However, the precise role of GSK-3α in cardiac mitophagy remains unknown. Herein, we investigated the role of GSK-3α in cardiac mitophagy by employing AC16 human cardiomyocytes under the condition of acute hypoxia. We observed that the gain-of-GSK-3α function profoundly induced mitophagy in the AC16 cardiomyocytes post-hypoxia. Moreover, GSK-3α overexpression led to increased ROS generation and mitochondrial dysfunction in cardiomyocytes, accompanied by enhanced mitophagy displayed by increased mt-mKeima intensity under hypoxia. Mechanistically, we identified that GSK-3α promotes mitophagy through upregulation of BNIP3, caused by GSK-3α-mediated increase in expression of HIF-1α and FOXO3a in cardiomyocytes post-hypoxia. Moreover, GSK-3α displayed a physical interaction with BNIP3 and, inhibited PINK1 and Parkin recruitment to mitochondria was observed specifically under hypoxia. Taken together, we identified a novel mechanism of mitophagy in human cardiomyocytes. GSK-3α promotes mitochondrial dysfunction and regulates FOXO3a -mediated BNIP3 overexpression in cardiomyocytes to facilitate mitophagy following hypoxia. An interaction between GSK-3α and BNIP3 suggests a role of GSK-3α in BNIP3 recruitment to the mitochondrial membrane where it enhances mitophagy in stressed cardiomyocytes independent of the PINK1/Parkin.
    Keywords:  BNIP3; FOXO3a; GSK-3alpha; Mitophagy; PINK1; Parkin
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2024.05.041
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